During the next funding period the research agenda will include a diverse set of investigations that are organized under a single primary goal - to improve the safety and stability of live attenuated cholera vaccines. Investigations will focus on the pathobiology of V. cholerae, the regulation of toxins and virulence factors discovered through genomic sequence analysis, and the mechanisms of horizontal transfer of virulence factor genes between strains of V. cholerae. Specifically, the investigators hope to define genes that are involved in the invasion (penetration) of mammalian HEp-2 cells by V cholerae, since this phenotype may correlate with the adverse symptoms (reactogenicity) caused by certain live cholera vaccines in human volunteer subjects. The specific mutations causing the motility defects in nonreactogenic, low invasive vaccine strains Peru-15 and Bengal-15 will also be defined by genetic methods and other defined mutations causing loss of bacterial motility will be characterized. Microarrays will be explored as a means of determining gene expression patterns across the entire V. cholerae genome for various types of regulatory mutants. The investigators will also continue analysis of the ToxR regulatory system focusing on interactions between ToxRS and TcpPH membrane proteins and the sodium pump encoded by the Nqr complex. Biochemical analysis will be performed on potential new toxins and virulence factors that have been revealed by genomic sequence analysis, microarray expression studies, and other genetic techniques. The mechanism of transfer of several genomic segments will be explored including a new phage corresponding to the TLC element, and the TCP and integron pathogenicity islands. The knowledge the investigators gain from these studies will be applied to the rationale construction and evaluation of new live, attenuated, V. cholerae vaccine candidates. The investigators will also work on the practical problem of how to prevent these vaccines strains from reacquiring the CTX phage or other genetic elements encoding virulence determinants. In an effort to accelerate functional genomics for V. cholerae, the investigators will also attempt to extend GAMBIT, a new method of genetic mapping, to V. cholerae.